An internationally standardized species identification test for use on suspected seized rhinoceros horn in the illegal wildlife trade

Abstract

Rhinoceros (rhino) numbers have dwindled substantially over the past century. As a result, three of the five species are now considered to be critically endangered, one species is vulnerable and one species is near-threatened. Poaching has increased dramatically over the past decade due to a growing demand for rhino horn products, primarily in Asia. Improved wildlife forensic techniques, such as validated tests for species identification of seized horns, are critical to aid current enforcement and prosecution efforts and provide a deterrent to future rhino horn trafficking. Here, we present an internationally standardized species identification test based on a 230 base pair cytochrome-b region. This test improves on previous nested PCR protocols and can be used for the discrimination of samples with <20pg of template DNA, thus suitable for DNA extracted from horn products. The assay was designed to amplify water buffalo samples, a common 'rhino horn' substitute, but to exclude human DNA, a common contaminant. Phylogenetic analyses using this partial cytochrome-b region resolved the five extant rhino species. Testing successfully returned a sequence and correct identification for all of the known rhino horn samples and vouchered rhino samples from museum and zoo collections, and provided species level identification for 47 out of 52 unknown samples from seizures. Validation and standardization was carried out across five different laboratories, in four different countries, demonstrating it to be an effective and reproducible test, robust to inter laboratory variation in equipment and consumables (such as PCR reagents). This is one of the first species identification tests to be internationally standardized to produce data for evidential proceedings and the first published validated test for rhinos, one of the flagship species groups of the illegal wildlife trade and for which forensic tools are urgently required. This study serves as a model for how species identification tests should be standardized and disseminated for wildlife forensic testing.

A rapid multiplex PCR assay for presumptive species identification of rhinoceros horns and its implementation in Vietnam

“…DNA-based wildlife forensics is one such measure that can be utilized in rhino horn trafficking investigations to confirm the presence of rhino horn and to identify the rhino species of origin. Due to the high price of horn, there has reportedly been many fake/substitute rhino horn products circulating the market, usually made from water buffalo horn, but these can also be made from other keratins, caseins, resins, wood, hair or plastics [ 4 , 6 ]. Therefore, the first aim in an investigation is to determine whether the seized product is real rhino horn [ 7 ]; and secondly, if the seized product is indeed rhino horn, to determine the species from which it originated.…”

“…DNA-based species identification protocols are often based on sequence differences of the cytochrome b (cyt b) gene [ 11 , 12 ]. Previous work validated a protocol that utilizes a 230 base pair (bp) region within cyt b and demonstrated that this gene region is appropriate to use for species identification of rhino horns [ 6 ]. However, in some laboratories, without in-house sequencing facilities and/or budgetary constraints, sequence-based methods can be expensive and time consuming.…”

Humans Are Animals, Too: Critical Commonalities and Differences Between Human and Wildlife Forensic Genetics

“…In general, validation techniques are very similar to those of human forensic science, though wildlife presents unique challenges, detailed below (also see Ref. ). Table highlights the differences between validation in human and wildlife forensics.…”

“…Protocols are written to reflect the validated range, as different species have different sequences at primer annealing locations, and may require varying reagent concentrations or thermal cycling parameters to produce good PCR products. A factor likely of lesser concern in human forensics is validation of markers to determine cross‐reactivity with other species ; in wildlife forensics, these tests should include common species which might be found in conjunction with the evidence (human, companion, and domesticated animals), common substitutes and look‐alike species, and close relatives of the target taxon .…”

Identification of rhinoceros keratin using direct analysis in real time time‐of‐flight mass spectrometry and multivariate statistical analysis

“…Genetic analyses of rhinoceros horn have proven successful for taxonomic identification, and even individualization . Such methods can be costly and time‐consuming, particularly when species‐ and individual‐level identifications are unnecessary.…”